Reran 20 ligands from VS1 (see below) but using ICM (Maestro ligprep was used for pH-ing) against 1U72. The following rank table was compiled:
Figure 12: ICM Protocol Results table (20 ligands vs. 1U72)
For reference, here is the VS1 results table using GOLD:
Figure 5: VS1 Ligand Docking Data into DHFR Active Site (From 7/16/13)
Here is the histogram of the scores ICM provided for me:
Figure 11: Score histogram for ICM run of 20 ligands vs. 1U72
7/30/13 VS4 Results
Ran verification library against NDM-1 Beta-Lactamase sample in GOLD using original protocol and redo protocol (specific protonation of histidines and removal of metal CONNECT records). Inserted results onto table. Do note Maestro ligprep was used to pH the library.
Name
Protein Code (PDB)
POS1
Faropenem
Penicillin
Clavulanic Acid
Aspirin
Original Ligand
REDO Pos1
REDO Faropenem
REDO Penicillin
REDO Clavulanic Acid
REDO Aspirin
REDO Original Ligand
Gabe H.
4HL2
115.04
80.56
76.95
65.54
66.96
86.35
113.25
85.00
68.53
70.53
69.24
91.88
Figure 10: VS4 Results Table (4HL2 vs. Verification Library)
Week 8
7/26/13 VS3 Results
Ran VS3 Protocol selecting 3QY7 (Tyrosine-protein phosphatase of Bacillus subtilis) using database CB1k_42. Controls were the original phosphate ligand, aspirin, pNPP, and two positive controls (known inhibitors). Table ranked by GOLD fitness score. 1st positive control has highest score in its three forms, however it does not fit Lipinki's rules. The top novel inhibitors all follow Lipinski's rules.
Figure 9: Best GOLD Ligand Dockings + Controls for 3QY7
Here are images for the dockings of the top performing control along with the 2 best novel inhibitors:
Figure 8: PyMol images for Positive Control and Best Novel Ligand GOLD Dockings on 3QY7
7/22/13 - 7/26/13 Protein Expression of FabI with Modified Expression Temperature and IPTG Levels
Continued work on expressing Daniel's target. This time, the incubation for expression conditions and IPTG levels were altered:
125uL IPTG @ 30degrees C (4-5 hours)
250uL IPTG @ 30degrees C (4-5 hours) 125uL IPTG @ 22degrees C (room temp) (overnight) 250uL IPTG @ 22degrees C (room temp) (overnight)
The room temperature incubation showed significant levels of the target protein in the solluble fraction of the cell following sonication. Work is now being done on purifying the protein.
7/23/13 VS2 Results
Ran VS 2 protocol. The following table shows results for docking onto 3DAT (bacterial):
Figure 8: Top Ligands for 3dat by GOLD fitness score
The following shows results for docking into 1u72 (human):
Figure 7: Top Ligands for 1u72 by GOLD fitness score
Week 7
7/17/13 - 7/19/13 Protein Expression of FabI with Modified Lysis Buffers
Began work on helping Daniel express his target. The issue we are dealing with is the protein not appearing in the soluble fraction of the cell after lysing and sonicating the culture. The following additives were applied to the lysis buffers in hopes of increasing the protein's solubility:
1. KCl 1M
2.MgCl2 .2M
3. Tween 80 .2% w/v
4.Tween 20 120uM
5.Sucrose 1M
6. Glycine 2%
7. Glycerol 40% v/v
Gel results showed no significant amount of protein in soluble fraction.
7/17/13 GFP + T7 Sequencing Results and Gel of GFP Plasmid
TCNTTTNNNNCATTTNNNTTNNNNNNNNNNNACNGNNN BLAST Search also failed on this sequence.
A gel was run on the whole plasmid to help understand results. It appears to contain contamination:
Figure 6: Agarose Gel of Cloned GFP. Lane 1: Skip. Lane 2: 1kb latter. Lane 2: GFP Plasmid.
7/16/13 VS1 Results
Followed VS1 Lab Protocol. The following is a table of the ligand docking results ranked by GOLD Fitness Score.
Figure 5: VS1 Ligand Docking Data into DHFR Active Site
Week 6
Gabe - good results. Post some virtual results (table) when you get some. - Dr. B 071713
DNA Sequencing Results for Purified GFP Plasmid after Transformation 7/12/13
GFP + M13Reverse: NNNNN It appears no DNA was found
GFP + M13Forward: NNNNNNNNNNNNNNNNNNANNNNNNTNCNTNNNANNAATANNNCNNNNTTNTTCNNNNNAANNCGGCNTTCTCNNNNNAGNNNTT NNNNNCNTTNNCGGGANNGNNTGNNNNNANTTTCNNNNCNGNCNNNNNANNNNTGNNNNNNNNNNCNNNANNNCCNCTCTNNNCN NTNGNNNNNCNNTGNTCNGNNNGGN The sequencing facility notified me that this sample is being rerun.
BLAST search was attempted on the GFP + M13 Forwards sample, but no results were found.
Performed transformation on cells with GFP plasmid. Performed midi prep to purify the DNA from the cell sample. Nanodrop results demonstrated the following:
Figure 4a: Nanodrop of purified GFP plasmid after midi prep.
RE Digest 6/25/13
RE Digest was done on pgbr22 plasmid using EcoRI, PvuII, and a combination of both. A gel was run with the following results (gel shared with Anita V.):
Figure 2: Shared Agarose Gel on RE Digest
Lane 1: Blank
Lane 2: DNA Ladder
Lane 3: Uncut Plasmid (Anita V.)
Lane 4: EcoRI HF + NE2 Buffer (Anita V.)
Lane 5: PvuII + NE2 Buffer (Anita V.)
Lane 6: EcoRI + PvuII + NE2 Buffer (Anita V.)
Lane 7: EcoRI + NEB Buffer (Anita V.)
Lane 8: PvuII + NE2 Buffer (Gabe H.)
Lane 9: PvuII + EcoRI HF + NE2 Buffer (Gabe H.)
Lane 10: EcoRI HF + NE2 Buffer (Gabe H.)
Virtual Gel on RE Digest on DNA Sequence Analysis 6/24/13
Figure 1: Gels and sequence on virtual digest of pgbr22
Week 9
8/1/13 ICM1 Results
Reran 20 ligands from VS1 (see below) but using ICM (Maestro ligprep was used for pH-ing) against 1U72. The following rank table was compiled:For reference, here is the VS1 results table using GOLD:
Here is the histogram of the scores ICM provided for me:7/30/13 VS4 Results
Ran verification library against NDM-1 Beta-Lactamase sample in GOLD using original protocol and redo protocol (specific protonation of histidines and removal of metal CONNECT records). Inserted results onto table. Do note Maestro ligprep was used to pH the library.Week 8
7/26/13 VS3 Results
Ran VS3 Protocol selecting 3QY7 (Tyrosine-protein phosphatase of Bacillus subtilis) using database CB1k_42.Controls were the original phosphate ligand, aspirin, pNPP, and two positive controls (known inhibitors). Table ranked by GOLD fitness score. 1st positive control has highest score in its three forms, however it does not fit Lipinki's rules. The top novel inhibitors all follow Lipinski's rules.
Here are images for the dockings of the top performing control along with the 2 best novel inhibitors:
7/22/13 - 7/26/13 Protein Expression of FabI with Modified Expression Temperature and IPTG Levels
Continued work on expressing Daniel's target. This time, the incubation for expression conditions and IPTG levels were altered:
125uL IPTG @ 30degrees C (4-5 hours)
250uL IPTG @ 30degrees C (4-5 hours)
125uL IPTG @ 22degrees C (room temp) (overnight)
250uL IPTG @ 22degrees C (room temp) (overnight)
The room temperature incubation showed significant levels of the target protein in the solluble fraction of the cell following sonication. Work is now being done on purifying the protein.
7/23/13 VS2 Results
Ran VS 2 protocol. The following table shows results for docking onto 3DAT (bacterial):
The following shows results for docking into 1u72 (human):
Week 7
7/17/13 - 7/19/13 Protein Expression of FabI with Modified Lysis Buffers
Began work on helping Daniel express his target. The issue we are dealing with is the protein not appearing in the soluble fraction of the cell after lysing and sonicating the culture. The following additives were applied to the lysis buffers in hopes of increasing the protein's solubility:1. KCl 1M
2.MgCl2 .2M
3. Tween 80 .2% w/v
4.Tween 20 120uM
5.Sucrose 1M
6. Glycine 2%
7. Glycerol 40% v/v
Gel results showed no significant amount of protein in soluble fraction.
7/17/13 GFP + T7 Sequencing Results and Gel of GFP Plasmid
Resequenced Cloned GFP with T7 Primer:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNCTTTCNTCNNGACGTTNCACACCNNTANGGTACGATCCAG
ATCNGGCTAGGCAGNCATTTTGAGNNAAGCGAGAACGCTAAACGANGGCAGAGCGNNAATCNCCCGNNNCCCATGCCCAC
ATCANGTTNNNTATTCANCATACCNNNNTNANTCAATGTTCCNNCCCACCAACGCATGCATTGACGATGTGGTTCTNAGA
NACTGCTTCATGGTCCANCTCATGCTATGNNNGGNACACAAGACNAAATCNNCTTTGANTGATCGAANAACTACCATCAC
TTANGTAAGCAGGGCGGGAAACCCACATTTTGCTTACACGATACAGGCCANAACAGATAACCGAAGANGNATGCCAGGTG
GAAGTTGTTGCGCTAGCTGGGACCCAACCACNCGNGAGANCNNNAAACNACTCNNNNNNNNTTTNNNNNNNNTCNANNNT
TNNNTNGGNNNGGGNTGGGGCGTNCTNCNTCCNNNNNNNAGGANTGNNNNNNNNCNNNNNNGTTGNNNGCCTGCATACAN
NNANACCNCNNCNACCNNACCCNNNTANNGGNNNNNNNNTNATCGNCNNCNNNGACNNCNNNNANNTCGAAGGACNNGAA
NNANCGNGCTTNNNNTNTACGACCNGNNNNNNNNNNACNTGANNCNACNNNCNNNANNNNGTNTNNANANTNNNGAGNGA
TNGACTNGNATNCTTNNTATNCNNNNNNTTNTNNCNNTATGNNNAAATNCNNCNTNANNNNNNTGNTCATGCNAGNGCTA
NGGGTTNTGGCCNTNNNNNNCNCCTTNNAGGNNAGNTCNCNGANNGNNGNNGCGGNNNTANNNGCTGGNANCCCGGNNTC
GGNGNNCGNGGANTGNTGAGCACNNNNNNNTGANGNTNGNNCGNTCNNNNGCTTGNGGCNTANNATCCANNNNATNAANC
TCNTTTNNNNCATTTNNNTTNNNNNNNNNNNACNGNNN
BLAST Search also failed on this sequence.
A gel was run on the whole plasmid to help understand results. It appears to contain contamination:
7/16/13 VS1 Results
Followed VS1 Lab Protocol. The following is a table of the ligand docking results ranked by GOLD Fitness Score.Week 6
Gabe - good results. Post some virtual results (table) when you get some. - Dr. B 071713DNA Sequencing Results for Purified GFP Plasmid after Transformation 7/12/13
GFP + M13Reverse:NNNNN
It appears no DNA was found
GFP + M13Forward:
NNNNNNNNNNNNNNNNNNANNNNNNTNCNTNNNANNAATANNNCNNNNTTNTTCNNNNNAANNCGGCNTTCTCNNNNNAGNNNTT
NNNNNCNTTNNCGGGANNGNNTGNNNNNANTTTCNNNNCNGNCNNNNNANNNNTGNNNNNNNNNNCNNNANNNCCNCTCTNNNCN
NTNGNNNNNCNNTGNTCNGNNNGGN
The sequencing facility notified me that this sample is being rerun.
BLAST search was attempted on the GFP + M13 Forwards sample, but no results were found.
Post-Transformation GFP Plasmid Midi Prep Purification 7/10/13
Performed transformation on cells with GFP plasmid. Performed midi prep to purify the DNA from the cell sample. Nanodrop results demonstrated the following:Avg. Results:
ng/ul: 79.35
Volume ul: 1200
260/280: 1.91
260/ 230: 4.15
yield (ng): 95220
yield (ug): 95
Week 5
PCR 7/3/13
Conducted PCR cloning on pgbr22Week 4
RE Digest 6/25/13RE Digest was done on pgbr22 plasmid using EcoRI, PvuII, and a combination of both. A gel was run with the following results (gel shared with Anita V.):
Lane 1: Blank
Lane 2: DNA Ladder
Lane 3: Uncut Plasmid (Anita V.)
Lane 4: EcoRI HF + NE2 Buffer (Anita V.)
Lane 5: PvuII + NE2 Buffer (Anita V.)
Lane 6: EcoRI + PvuII + NE2 Buffer (Anita V.)
Lane 7: EcoRI + NEB Buffer (Anita V.)
Lane 8: PvuII + NE2 Buffer (Gabe H.)
Lane 9: PvuII + EcoRI HF + NE2 Buffer (Gabe H.)
Lane 10: EcoRI HF + NE2 Buffer (Gabe H.)
Virtual Gel on RE Digest on DNA Sequence Analysis 6/24/13